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Institute
- Graduate School of Life Sciences (28)
- Theodor-Boveri-Institut für Biowissenschaften (13)
- Physikalisches Institut (7)
- Institut für Informatik (6)
- Institut für Mathematik (6)
- Institut für Psychologie (6)
- Institut für Pharmazie und Lebensmittelchemie (5)
- Institut für Theoretische Physik und Astrophysik (5)
- Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde (3)
- Fakultät für Chemie und Pharmazie (3)
Sonstige beteiligte Institutionen
G-quadruplex structures are highly stable alternative DNA structures that can, when not properly regulated, impede replication fork progression and cause genome instability (Castillo Bosch et al, 2014; Crabbe et al, 2004; Koole et al, 2014; Kruisselbrink et al, 2008; London et al, 2008; Lopes et al, 2011; Paeschke et al, 2013; Paeschke et al, 2011; Piazza et al, 2015; Piazza et al, 2010; Piazza et al, 2012; Ribeyre et al, 2009; Sabouri et al, 2014; Sarkies et al, 2012; Sarkies et al, 2010; Schiavone et al, 2014; Wu & Spies, 2016; Zimmer et al, 2016). The aim of this thesis was to identify novel G-quadruplex interacting proteins in Saccharomyces cerevisiae and to unravel their regulatory function at these structures to maintain genome integrity. Mms1 and Rtt101 were identified as G-quadruplex binding proteins in vitro via a pull-down experiment with subsequent mass spectrometry analysis. Rtt101, Mms1 and Mms22, which are all components of an ubiquitin ligase (Rtt101Mms1/Mms22), are important for the progression of the replication fork following fork stalling (Luke et al, 2006; Vaisica et al, 2011; Zaidi et al, 2008). The in vivo binding of endogenously tagged Mms1 to its target regions was analyzed genome-wide using chromatin-immunoprecipitation followed by deep-sequencing. Interestingly, Mms1 bound independently of Mms22 and Rtt101 to G-rich regions that have the potential to form G-quadruplex structures. In vitro, formation of G-quadruplex structures could be shown for the G-rich regions Mms1 bound to. This binding was observed throughout the cell cycle. Furthermore, the deletion of MMS1 caused replication fork stalling as evidenced by increased association of DNA Polymerase 2 at Mms1 dependent sites. A gross chromosomal rearrangement assay revealed that deletion of MMS1 results in a significantly increased genome instability at G-quadruplex motifs compared to G-rich or non-G-rich regions. Additionally, binding of the helicase Pif1, which unwinds G4 structures in vitro (Paeschke et al, 2013; Ribeyre et al, 2009; Sanders, 2010; Wallgren et al, 2016), to Mms1 binding sites was reduced in mms1 cells. The data presented in this thesis, together with published data, suggests a novel mechanistic model in which Mms1 binds to G-quadruplex structures and enables Pif1 association. This allows for replication fork progression and genome integrity.
Biological systems such as cells or whole organisms are governed by complex regulatory networks of transcription factors, hormones and other regulators which determine the behavior of the system depending on internal and external stimuli. In mathematical models of these networks, genes are represented by interacting “nodes” whose “value” represents the activity of the gene.
Control processes in these regulatory networks are challenging to elucidate and quantify. Previous control centrality metrics, which aim to mathematically capture the ability of individual nodes to control biological systems, have been found to suffer from problems regarding biological plausibility.
This thesis presents a new approach to control centrality in biological networks. Three types of network control are distinguished: Total control centrality quantifies the impact of gene mutations and identifies potential pharmacological targets such as genes involved in oncogenesis (e.g. zinc finger protein GLI2 or bone morphogenetic proteins in chondrocytes). Dynamic control centrality describes relaying functions as observed in signaling cascades (e.g control in mouse colon stem cells). Value control centrality measures the direct influence of the value of the node on the network (e.g. Indian hedgehog as an essential regulator of proliferation in chondrocytes). Well-defined network manipulations define all three centralities not only for nodes, but also for the interactions between them, enabling detailed insights into network pathways.
The calculation of the new metrics is made possible by substantial computational improvements in the simulation algorithms for several widely used mathematical modeling paradigms for genetic regulatory networks, which are implemented in the regulatory network simulation framework Jimena created for this thesis.
Applying the new metrics to biological networks and artificial random networks shows how these mathematical concepts correspond to experimentally verified gene functions and signaling pathways in immunity and cell differentiation. In contrast to controversial previous results even from the Barabási group, all results indicate that the ability to control biological networks resides in only few driver nodes characterized by a high number of connections to the rest of the network. Autoregulatory loops strongly increase the controllability of the network, i.e. its ability to control itself, and biological networks are characterized by high controllability in conjunction with high robustness against mutations, a combination that can be achieved best in sparsely connected networks with densities (i.e. connections to nodes ratios) around 2.0 - 3.0.
The new concepts are thus considerably narrowing the gap between network science and biology and can be used in various areas such as system modeling, plausibility trials and system analyses.
Medical applications discussed in this thesis include the search for oncogenes and pharmacological targets, as well their functional characterization.
Biochemical and molecular characterization of an original master sex determining gene in Salmonids
(2016)
Sexual development is a fundamental and versatile process that shapes animal morphology, physiology and behavior. The underlying developmental process is composed of the sex determination and the sex differentiation. Sex determination mechanisms are extremely labile among taxa. The initial triggers of the sex determination process are often genetics called sex determining genes. These genes are expressed in the bipotential gonad and tilt the balance to a developmental program allowing the differentiation of either a testis or an ovary. Fish represent a large and fascinating vertebrate group to study both sex determination and sex differentiation mechanisms. To date, among the known sex determining genes, three gene families namely sox, dmrt and TGF-β factors govern this developmental program. As exception to this rule, sdY “sexually dimorphic on the Y” does not belong to one of these families as it comes from the duplication / evolution of an ancestor gene related to immunity, i.e., the interferon related factor 9, irf9. sdY is the master sex determining gene in salmonids, a group of fishes that include species such as rainbow trout and Atlantic salmon. The present study was aimed to firstly characterize the features of SdY protein. Results indicate that SdY is predominantly localized in the cytoplasm tested in various fish and mammalian cell lines and confirmed by different methods. Predictive in silico analysis revealed that SdY is composed of a β-sandwich core surrounded by three α-helices as well specific characteristics conferring a putative protein-protein interaction site. Secondly, the study was aimed to understand how SdY could trigger testicular differentiation. SdY is a truncated divergent version of Irf9 that has a conserved protein-protein domain but lost the DNA interaction domain of its ancestor gene. It was then hypothesized that SdY could initiate testicular differentiation by protein-protein interactions. To evaluate this we first conducted a yeast-two-hybrid screen that revealed a high proportion of transcription factors including fox proteins. Using various biochemical and cellular methods we confirm an interaction between SdY and Foxl2, a major transcription factor involved in ovarian differentiation and identity maintenance. Interestingly, the interaction of SdY with Foxl2 leads to nuclear translocation of SdY from the cytoplasm. Furthermore, this SdY translocation mechanism was found to be specific to fish Foxl2 and to a lesser extend Foxl3 and not other Fox proteins or mammalian FoxL2. In addition, we found that this interaction allows the stabilization of SdY and prevents its degradation. Finally, to better decipher SdY action we used as a model a mutated version of SdY that was identified in XY females of Chinook salmon natural population. Results show that this mutation induces a local conformation defect obviously leading to a misfolded protein and a quick degradation. Moreover, the mutated version compromised the interaction with Foxl2 defining a minimal threshold to induce testicular differentiation. Altogether results from my thesis propose that SdY would trigger testicular differentiation in salmonids by preventing Foxl2 to promote ovarian differentiation. Further research should be now carried out on how this interaction of SdY and Foxl2 acts in-vivo.
MYC is a transcription factor, whose expression is elevated or deregulated in many human cancers (up to 70%) and is often associated with aggressive and poorly differentiated tumors. Although MYC is extensively studied, discrepancies have emerged about how this transcription factor works. In primary lymphocytes, MYC promotes transcriptional amplification of virtually all genes with an open promoter, whereas in tumor cells MYC regulates specific sets of genes that have significant prognostic value. Furthermore, the set of target genes that distinguish MYC’s physiological function from the pathological/oncogenic one, whether it exists or not, has not been fully understood yet.
In this study, it could be shown that MYC protein levels within a cell and promoter affinity (determined by E-box presence or interaction with other proteins) of target genes toward MYC are important factors that influence MYC activity. At low levels, MYC can amplify a certain transcriptional program, which includes high affinity binding sites, whereas at high levels MYC leads to the specific up- and down regulation of genes with low affinity. Moreover, the promoter affinity characterizes different sets of target genes which can be distinguished in the physiological or oncogenic MYC signatures.
MYC-mediated repression requires higher MYC levels than activation and formation of a complex with MIZ1 is necessary for inhibiting expression of a subset of MYC target genes.
Novel Approaches to Antimicrobial Therapy of Pneumonia using Antibiotics and Therapeutic Antibodies
(2016)
Nosocomial pneumonia is mostly caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the standard antibiotic therapy is affected by increasing emergence of bacterial resistance. Therefore, novel therapeutic options are in high demand. New antimicrobial agents alone cannot handle the problem of increasing bacterial resistance but innovative drug delivery strategies and fast identification of infection causing pathogens are required to diminish bacterial resistance development. A very promising approach to improve the therapy of pneumonia is presented by local drug delivery to the lung. This application method enables high local drug concentrations in the lung leading to shorter application of antibiotics and hence reduces the risk of resistance development. Furthermore, the systemic concentration is lowered reducing the emergence of adverse effects.
Therefore, in this thesis several approaches to improve the therapy of MRSA pneumonia are studied.
One approach to achieve an efficient local delivery of antibiotics are nano-sized drug delivery systems which enable the nebulization of poorly-soluble antibiotics and can lead to even higher local drug concentrations due to their small size since nanoparticles improve mucus penetration and decrease phagocytosis by alveolar macrophages. Here, an analytical setup was developed that facilitates the identification of optimal preparation conditions for drug polyelectrolyte nanoplexes.
Another promising approach to support antimicrobial therapy of pneumonia is presented by antibody-based immunotherapy. Since the stability of the antibody and hence its therapeutic activity are endangered during production, transport, storage, and application, a stabilizing formulation was developed for hUK-66, an antibody targeting surface antigens of S. aureus. Furthermore, nebulization of this formulated monoclonal antibody was studied to enable local application. Finally, the immunotherapeutic efficacy of the nebulized hUK-66 formulation was investigated in an animal in vivo study.
Furthermore, rapid identification of the infection triggering pathogen is very important. The selective detection of S. aureus was achieved using optical planar Bragg grating sensors functionalized with hUK-66. In addition, the reusability of this system was studied applying a surface functionalization based on the cross-linker SPDP which enables a reversible fixation of the antibody.
Identifying novel driver genes in cancer remains a crucial step towards development of new therapeutic approaches and the basic understanding of the disease.
This work describes the impact of the AP1 transcription activator component FOSL1 on melanoma maintenance. FOSL1 is strongly upregulated during the progression of melanoma and the protein abundance is highest in metastases. I found that the regulation of FOSL1 is strongly dependent on ERK1/2- and PI3K- signaling, two pathways frequently activated in melanoma. Moreover, the involvement of p53 in FOSL1 regulation in melanoma was investigated. Elevated levels of the tumor suppressor led to decreased FOSL1 protein levels in a miR34a/miR34c- dependent manner.
The benefit of elevated FOSL1 amounts in human melanoma cell lines was analyzed by overexpression of FOSL1 in cell lines with low endogenous FOSL1 levels. Enhanced levels of FOSL1 had several pro-tumorigenic effects in human melanoma cell lines. Besides increased proliferation and migration rates, FOSL1 overexpression induced the colony forming ability of the cells. Additionally, FOSL1 was necessary for anchorage independent growth in 3D cell cultures. Microarray analyses revealed novel downstream effectors of FOSL1. On the one hand, FOSL1 was able to induce the transcription of different neuron-related genes, such as NEFL, NRP1 and TUBB3. On the other hand, FOSL1 influenced the transcription of DCT, a melanocyte specific gene, in dependence of the differentiation of the melanoma cell line, indicating dedifferentiation.
Furthermore, FOSL1 induced the transcription of HMGA1, a chromatin remodeling protein with reprogramming ability, which is characteristic for stem cells. Consequently, the influence of HMGA1 on melanoma maintenance was investigated. In addition to decreased proliferation and reduced anoikis resistance, HMGA1 knockdown reduced melanoma cell survival. Interestingly, the FOSL1 induced pro-tumorigenic effects were demonstrated to be dependent on the HMGA1 level. HMGA1 manipulation reversed FOSL1 induced proliferation and colony forming ability, as well as the anchorage independent growth effect.
In conclusion, I could show that additional FOSL1 confers a clear growth benefit to melanoma cells. This benefit is attributed to the induction of stem cell determinants, but can be blocked by the inhibition of the ERK1/2 or PI3K signaling pathways.
A novel growth method has been developed, allowing for the growth of strained HgTe shells on CdTe nanowires (NWs). The growth of CdTe-HgTe core-shell NWs required high attention in controlling basic parameters like substrate temperature and the intensity of supplied material fluxes. The difficulties in finding optimized growth conditions have been successfully overcome in this work.
We found the lateral redistribution of liquid growth seeds with a ZnTe growth start to be crucial to trigger vertical CdTe NW growth. Single crystalline zinc blende CdTe NWs grew, oriented along [111]B. The substrate temperature was the most critical parameter to achieve straight and long wires. In order to adjust it, the growth was monitored by reflection high-energy electron diffraction, which was used for fine tuning of the temperature over time in each growth run individually. For optimized growth conditions, a periodic diffraction pattern allowed for the detailed analysis of atomic arrangement on the surfaces and in the bulk. The ability to do so reflected the high crystal quality and ensemble uniformity of our CdTe NWs. The NW sides were formed by twelve stable, low-index crystalline facets. We observed two types stepped and polar sides, separated by in total six flat and non-polar facets.
The high crystalline quality of the cores allowed to grow epitaxial HgTe shells around. We reported on two different heterostructure geometries. In the first one, the CdTe NWs exhibit a closed HgTe shell, while for the second one, the CdTe NWs are overgrown mainly on one side. Scanning electron microscopy and scanning transmission electron microscopy confirmed, that many of the core-shell NWs are single crystalline zinc blende and have a high uniformity. The symmetry of the zinc blende unit cell was reduced by residual lattice strain. We used high-resolution X-ray diffraction to reveal the strain level caused by the small lattice mismatch in the heterostructures. Shear strain has been induced by the stepped hetero-interface, thereby stretching the lattice of the HgTe shell by 0.06 % along a direction oriented with an angle of 35 ° to the interface.
The different heterostructures obtained, were the base for further investigation of quasi-one-dimensional crystallites of HgTe. We therefore developed methods to reliably manipulate, align, localize and contact individual NWs, in order to characterize the charge transport in our samples. Bare CdTe cores were insulating, while the HgTe shells were conducting. At low temperature we found the mean free path of charge carriers to be smaller, but the phase coherence length to be larger than the sample size of several hundred nanometers. We observed universal conductance fluctuations and therefore drew the conclusion, that the trajectories of charge carriers are defined by elastic backscattering at randomly distributed scattering sites. When contacted with superconducting leads, we saw induced superconductivity, multiple Andreev reflections and the associated excess current. Thus, we achieved HgTe/superconductor interfaces with high interfacial transparency.
In addition, we reported on the appearance of peaks in differential resistance at Delta/e for HgTe-NW/superconductor and 2*Delta/e for superconductor/HgTe-NW/superconductor junctions, which is possibly related to unconventional pairing at the HgTe/superconductor interface. We noticed that the great advantage of our self-organized growth is the possibility to employ the metallic droplet, formerly seeding the NW growth, as a superconducting contact. The insulating wire cores with a metallic droplet at the tip have been overgrown with HgTe in a fully in-situ process. A very high interface quality was achieved in this case.
Wilms tumor protein 1 (WT1) is a suitable target to develop an immunotherapeutic approach against high risk acute myeloid leukemia (AML), particularly their relapse after allogeneic hematopoietic stem cell transplantation (HSCT). As an intracellular protein traversing between nucleus and cytoplasm, recombinant expression of WT1 is difficult. Therefore, an induction of WT1-specific T-cell responses is mostly based on peptide vaccination as well as dendritic cell (DC) electroporation with mRNA encoding full-length protein to mount WT1-derived peptide variations presented to T cells. Alternatively, the WT1 peptide presentation could be broadened by forcing receptor-mediated endocytosis of DCs.
In this study, antibody fusion proteins consisting of an antibody specific to the human DEC205 endocytic receptor and various fragments of WT1 (anti-hDEC205-WT1) were generated for a potential DC-targeted recombinant WT1 vaccine. Anti-hDEC205-WT1 antibody fusion proteins containing full-length or major parts of WT1 were not efficiently expressed and secreted due to their poor solubility and secretory capacity. However, small fragment-containing variants: anti-hDEC205-WT110-35, anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were obtained in good yields.
Since three of these fusion proteins contain the most of the known immunogenic epitopes in their sequences, the anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were tested for their T-cell stimulatory capacities. Mature monocyte-derived DCs loaded with anti-hDEC205-WT191-138 could induce ex vivo T-cell responses in 12 of 16 blood samples collected from either healthy or HSC transplanted individuals compared to included controls (P < 0.01). Furthermore, these T cells could kill WT1-overexpressing THP-1 leukemia cells in vitro after expansion.
In conclusion, alongside proving the difficulty in expression and purification of intracellular WT1 as a vaccine protein, our results from this work introduce an alternative therapeutic vaccine approach to improve an anti-leukemia immune response in the context of allogeneic HSCT and potentially beyond.
High-Resolution X-ray Imaging based on a Liquid-Metal-Jet-Source with and without X-ray Optics
(2016)
With increasing miniaturization in industry and medical technology, non-destructive testing techniques are an area of everincreasing importance. In this framework, X-ray microscopy offers an efficient tool for the analysis, understanding and quality assurance of microscopic species, in particular as it allows reconstructing three-dimensional data sets of the whole sample’s volumevia computed tomography (CT).
The following thesis describes the conceptualization, design, construction and characterization of a compact laboratory-based X-ray microscope in the hard X-ray regime around 9 keV, corresponding to a wavelength of 0.134 nm. Hereby, the main focus is on the optimization of resolution and contrast at relatively short exposure times. For this, a novel liquid-metal-jet anode source is the basis. Such only recently commercially available X-ray source reaches a higher brightness than other conventional laboratory sources, i.e. the number of emitted photons (X-ray quanta) per area and solid angle is exceptionally high. This is important in order to reach low exposure times. The reason for such high brightness is the usage of the rapidly renewing anode out of liquid metal which enables an effective dissipation of heat, normally limiting the creation of high intensities on a small area.
In order to cover a broad range of different samples, the microscope can be operated in two
modes. In the “micro-CT mode”, small pixels are realized with a crystal-scintillator and an
optical microscope via shadow projection geometry. Therefore, the resolution is limited by the emitted wavelength of the scintillator, as well as the blurring of the screen. However, samples in the millimeter range can be scanned routinely with low exposure times. Additionally, this mode is optimized with respect to in-line phase contrast, where edges of an object are enhanced and thus better visible.
In the second “nano-CT mode”, a higher resolution can be reached via X-ray lenses. However,
their production process is due to the physical properties of the hard X-ray range - namely high absorption and low diffraction - extremely difficult, leading typically to low performances. In combination with a low brightness, this leads to long exposure times and high requirements in terms of stability, which is one of the key problems of laboratory-based X-ray microscopy. With the here-developed setup and the high brightness of its source, structures down to 150 nm are resolved at moderate exposure times (several minutes per image) and nano-CTs can be obtained.
Affective states in the context of learning and achievement can influence the learning process essentially. The impact of affective states can be both directly on the learning performance and indirectly mediated via, for example, motivational processes. Positive activating affect is often associated with increased memory skills as well as advantages in creative problem solving. Negative activating affect on the other hand is regarded to impair learning outcomes because of promoting task-irrelevant thinking. While these relationships were found to be relatively stable in correlation studies, causal relationships have been examined rarely so far. This dissertation aims to investigate the effects of positive and negative affective states in multimedia learning settings and to identify potential moderating factors. Therefore, three experimental empirical studies on university students were conducted. In Experiment 1, N = 57 university students were randomly allocated to either a positive or negative affect induction group. Affects were elicited using short film clips. After a 20-minute learning phase in a hypertext-based multimedia learning environment on “functional neuroanatomy” the learners’ knowledge as well as transfer performance were measured. It was assumed that inducing positive activating affect should enhance learning performance. Eliciting negative activating affect on the other hand should impair learning performance. However, it was found that the induction of negative activating affect prior to the learning phase resulted in slight deteriorations in knowledge. Contrary to the assumptions, inducing positive activating affect before the learning phase did not improve learning performance. Experiment 2 induced positive activating affect directly during learning. To induce affective states during the entire duration of the learning phase, Experiment 2 used an emotional design paradigm. Therefore, N = 111 university students were randomly assigned to learn either in an affect inducing multimedia learning environment (use of warm colours and round shapes) or an affectively neutral counterpart (using shades of grey and angular shapes) on the same topic as in Experiment 1. Again, knowledge as well as transfer performance were measured after learning for 20 minutes. In addition, positive and negative affective states were measured before and after learning. Complex interaction patterns between the treatment and initial affective states were found. Specifically, learners with high levels of positive affect before learning showed better transfer performance when they learned in the affect inducing learning environment. Regarding knowledge, those participants who reported high levels of negative activating affect prior to the learning period performed worse. However, the effect on knowledge did not occur for those students learning in the affect inducing learning environment. For knowledge, the treatment therefore protected against poorer performance due to high levels of negative affective states. Results of Experiment 2 showed that the induction of positive activating affect influenced learning performance positively when taking into account affective states prior to the learning phase. In order to confirm these interaction effects, a conceptual replication of the previous experiment was conducted in Experiment 3. Experiment 3 largely retained the former study design, but changed the learning materials and tests used. Analogous to Experiment 2, N = 145 university students learning for 20 minutes in either an affect inducing or an affectively neutral multimedia learning environment on “eukaryotic cell”. To strengthen the treatment, Experiment 3 also used anthropomorphic design elements to induce affective states next to warm colours and round shapes. Moreover, in order to assess the change in affective states more exactly, an additional measurement of positive and negative affective states after half of the learning time was inserted. Knowledge and transfer were assessed again to measure learning performance. The learners’ memory skills were used as an additional learning outcome. To control the influence of potential confounding variables, the participants’ general and current achievement motivation as well as interest, and emotion regulation skills were measured. Contrary to the assumptions, Experiment 3 could not confirm the interaction effects of Experiment 2. Instead, there was a significant impact of positive activating affect prior to the learning phase on transfer, irrespective of the learners’ group affiliation. This effect was further independent of the control variables that were measured. Nevertheless, the results of Experiment 3 fit into the picture of findings regarding “emotional design” in hypermedia learning settings. To date, the few publications that have used this approach propose heterogeneous results, even when using identical materials and procedures.
Beyond the state of the art, towards intuitive and reliable non-visual Brain-Computer-Interfacing
(2016)
For the present work three main goals were formulated:
goal 1 To design a tactile BCI used for mobility which is
intuitive (G1.1), reliable and fast while being usable
by participants aged 50 years and above.
goal 2 To design an auditory BCI used for communication
which is intuitive and reliable.
goal 3 To examine the effects of training on tactile and
auditory BCI performance.
Three studies were performed to achieve these goals.
In the first study nine participants aged above 50 years
performed a five-session training after which eight participants
were able to navigate a virtual wheelchair with
mean accuracy above 95% and an ITR above 20 bits / min.
In the second study 15 participants, four of them endusers
with motor-impairment, were able to communicate
meaningful with high accuracies using an auditory BCI.
In the third study nine healthy and nine visually impaired
participants (regarded as sensory experts for non-visual
perception) performed tactile, auditory and visual (for
healthy participants only) copy tasks. Participants with
trained perception significantly outperformed control
participants for tactile but not for auditory performance.
Tactile performance of sensory experts was on equal levels
as the visual performance of control participants.
We were able to demonstrate viability of intuitive gazeindependent
tactile and auditory BCI. Our tactile BCI performed
on levels similar to those of visual BCI, outperforming
current tactile BCI protocols. Furthermore, we were
able to demonstrate significant beneficial effect of training
on tactile BCI performance. Our results demonstrate previously
untapped potential for tactile BCI and avenues for
future research in the field of gaze-independent BCI.
This doctoral thesis is concerned with the mathematical modeling of magnetoelastic materials and the analysis of PDE systems describing these materials and obtained from a variational approach.
The purpose is to capture the behavior of elastic particles that are not only magnetic but exhibit a magnetic domain structure which is well described by the micromagnetic energy and the Landau-Lifshitz-Gilbert equation of the magnetization. The equation of motion for the material’s velocity is derived in a continuum mechanical setting from an energy ansatz. In the modeling process, the focus is on the interplay between Lagrangian and Eulerian coordinate systems to combine elasticity and magnetism in one model without the assumption of small deformations.
The resulting general PDE system is simplified using special assumptions. Existence of weak solutions is proved for two variants of the PDE system, one including gradient flow dynamics on the magnetization, and the other featuring the Landau-Lifshitz-Gilbert equation. The proof is based on a Galerkin method and a fixed point argument. The analysis of the PDE system with the Landau-Lifshitz-Gilbert equation uses a more involved approach to obtain weak solutions based on G. Carbou and P. Fabrie 2001.
Eclosion is the emergence of an adult insect from the pupal case at the end of development. In the fruit fly Drosophila melanogaster, eclosion is a circadian clock-gated event and is regulated by various peptides. When studied on the population level, eclosion reveals a clear rhythmicity with a peak at the beginning of the light-phase that persists also under constant conditions. It is a long standing hypothesis that eclosion gating to the morning hours with more humid conditions is an adaption to reduce water loss and increase the survival. Eclosion behavior, including the motor pattern required for the fly to hatch out of the puparium, is orchestrated by a well-characterized cascade of peptides. The main components are ecdysis-triggering hormone (ETH), eclosion hormone (EH) and crustacean cardioactive peptide (CCAP). The molt is initiated by a peak level and pupal ecdysis by a subsequent decline of the ecdysteroid ecdysone. Ecdysteroids are produced by the prothoracic gland (PG), an endocrine tissue that contains a peripheral clock and degenerates shortly after eclosion. Production and release of ecdysteroids are regulated by the prothoracicotropic hormone (PTTH).
Although many aspects of the circadian clock and the peptidergic control of the eclosion behavior are known, it still remains unclear how both systems are interconnected. The aim of this dissertation research was to dissect this connection and evaluate the importance of different Zeitgebers on eclosion rhythmicity under natural conditions.
Potential interactions between the central clock and the peptides regulating ecdysis motor behavior were evaluated by analyzing the influence of CCAP on eclosion rhythmicity. Ablation and silencing of CCAP neurons, as well as CCAP null-mutation did not affect eclosion rhythmicity under either light or temperature entrainment nor under natural conditions.
To dissect the connection between the central and the peripheral clock, PTTH neurons were ablated. Monitoring eclosion under light and temperature entrainment revealed that eclosion became arrhythmic under constant conditions. However, qPCR expression analysis revealed no evidence for cycling of Ptth mRNA in pharate flies. To test for a connection with pigment-dispersing factor (PDF)-expressing neurons, the PDF receptor (PDFR) and short neuropeptide F receptor (sNPFR) were knocked down in the PTTH neurons. Knockdown of sNPFR, but not PDFR, resulted in arrhythmic eclosion under constant darkness conditions. PCR analysis of the PTTH receptor, Torso, revealed its expression in the PG and the gonads, but not in the brain or eyes, of pharate flies. Knockdown of torso in the PG lead to arrhythmicity under constant conditions, which provides strong evidence for the specific effect of PTTH on the PG. These results suggest connections from the PDF positive lateral neurons to the PTTH neurons via sNPF signaling, and to the PG via PTTH and Torso. This interaction presumably couples the period of the peripheral clock in the PG to that of the central clock in the brain.
To identify a starting signal for eclosion and possible further candidates in the regulation of eclosion behavior, chemically defined peptidergic and aminergic neurons were optogenetically activated in pharate pupae via ChR2-XXL. This screen approach revealed two candidates for the regulation of eclosion behavior: Dromyosuppressin (DMS) and myo-inhibitory peptides (MIP). However, ablation of DMS neurons did not affect eclosion rhythmicity or success and the exact function of MIP must be evaluated in future studies.
To assess the importance of the clock and of possible Zeitgebers in nature, eclosion of the wildtype Canton S and the clock mutant per01 and the PDF signaling mutants pdf01 and han5304 was monitored under natural conditions. For this purpose, the Würzburg eclosion monitor (WEclMon) was developed, which is a new open monitoring system that allows direct exposure of pupae to the environment. A general decline of rhythmicity under natural conditions compared to laboratory conditions was observed in all tested strains. While the wildtype and the pdf01 and han5304 mutants stayed weakly rhythmic, the per01 mutant flies eclosed mostly arrhythmic. PDF and its receptor (PDFR encoded by han) are required for the synchronization of the clock network and functional loss can obviously be compensated by a persisting synchronization to external Zeitgebers. The loss of the central clock protein PER, however, lead to a non-functional clock and revealed the absolute importance of the clock for eclosion rhythmicity. To quantitatively analyze the effect of the clock and abiotic factors on eclosion rhythmicity, a statistical model was developed in cooperation with Oliver Mitesser and Thomas Hovestadt. The modelling results confirmed the clock as the most important factor for eclosion rhythmicity. Moreover, temperature was found to have the strongest effect on the actual shape of the daily emergence pattern, while light has only minor effects. Relative humidity could be excluded as Zeitgeber for eclosion and therefore was not further analyzed.
Taken together, the present dissertation identified the so far unknown connection between the central and peripheral clock regulating eclosion. Furthermore, a new method for the analysis of eclosion rhythms under natural conditions was established and the necessity of a functional clock for rhythmic eclosion even in the presence of multiple Zeitgebers was shown.
Staphylococcus aureus is a prevalent commensal bacterium which represents one of the leading causes in health care-associated bacterial infections worldwide and can cause a variety of different diseases ranging from simple abscesses to severe and life threatening infections including pneumonia, osteomyelitis and sepsis.
In recent times multi-resistant strains have emerged, causing severe problems in nosocomial as well as community-acquired (CA) infection settings, especially in the United States (USA). Therefore S. aureus has been termed as a superbug by the WHO, underlining the severe health risk originating from it. Today, infections in the USA are dominated by S. aureus genotypes which are classified as USA300 and USA400, respectively. Strains of genotype USA300 are responsible for about 70% of the CA infections.
The molecular mechanisms which render S. aureus such an effective pathogen are still not understood in its entirety. For decades S. aureus was thought to be a strictly extracellular pathogen relying on pore-forming toxins like α-hemolysin to damage human cells and tissue. Only recently it has been shown that S. aureus can enter non-professional phagocytes, using adhesins like the fibronectin-binding proteins which mediate an endocytotic uptake into the host cells. The bacteria are consequently localized to endosomes, where the degradation of enclosed bacterial cells through phagosome maturation would eventually occur.
S. aureus can avoid degradation, and translocate to the cellular cytoplasm, where it can replicate. The ability to cause this so-called phagosomal escape has mainly been attributed to a family of amphiphilic peptides called phenol soluble modulins (PSMs), but as studies have shown, they are not sufficient.
In this work I used a transposon mutant library in combination with automated fluorescence microscopy to screen for genes involved in the phagosomal escape process and intracellular survival of S. aureus. I thereby identified a number of genes, including a non-ribosomal peptide synthetase (NRPS). The NRPS, encoded by the genes ausA and ausB, produces two types of small peptides, phevalin and tyrvalin. Mutations in the ausAB genes lead to a drastic decrease in phagosomal escape rates in epithelial cells, which were readily restored by genetic complementation in trans as well as by supplementation of synthetic phevalin. In leukocytes, phevalin interferes with calcium fluxes and activation of neutrophils and promotes cytotoxicity of intracellular bacteria in both, macrophages and neutrophils. Further ausAB is involved in survival and virulence of the bacterium during mouse lung pneumoniae.
The here presented data demonstrates the contribution of the bacterial cyclic dipeptide phevalin to S. aureus virulence and suggests, that phevalin directly acts on a host cell target to promote cytotoxicity of intracellular bacteria.
A large fraction of human tumors exhibits aberrant expression of the oncoprotein MYC. As a transcription factor regulating various cellular processes, MYC is also crucially involved in normal development. Direct targeting of MYC has been a major challenge for molecular cancer drug discovery. The proof of principle that its inhibition is nevertheless feasible came from in vivo studies using a dominant-negative allele of MYC termed OmoMYC. Systemic expression of OmoMYC triggered long-term tumor regression with mild and fully reversible side effects on normal tissues.
In this study, OmoMYC’s mode of action was investigated combining methods of structural biology and functional genomics to elucidate how it is able to preferentially affect oncogenic functions of MYC.
The crystal structure of the OmoMYC homodimer, both in the free and the E-box-bound state, was determined, which revealed that OmoMYC forms a stable homodimer, and as such, recognizes DNA via the same base-specific DNA contacts as the MYC/MAX heterodimer. OmoMYC binds DNA with an equally high affinity as MYC/MAX complexes. RNA-sequencing showed that OmoMYC blunts both MYC-dependent transcriptional activation and repression. Genome-wide DNA-binding studies using chromatin immunoprecipitation followed by high-throughput sequencing revealed that OmoMYC competes with MYC/MAX complexes on chromatin, thereby reducing their occupancy at consensus DNA binding sites. The most prominent decrease in MYC binding was seen at low-affinity promoters, which were invaded by MYC at oncogenic levels. Strikingly, gene set enrichment analyses using OmoMYC-regulated genes enabled the identification of tumor subgroups with high MYC levels in multiple tumor entities. Together with a targeted shRNA screen, this identified novel targets for the eradication of MYC-driven tumors, such as ATAD3A, BOP1, and ADRM1.
In summary, the findings suggest that OmoMYC specifically inhibits tumor cell growth by attenuating the expression of rate-limiting proteins in cellular processes that respond to elevated levels of MYC protein using a DNA-competitive mechanism. This opens up novel strategies to target oncogenic MYC functions for tumor therapy.
Graphene-based single-electron and hybrid devices, their lithography, and their transport properties
(2016)
This work explores three different aspects of graphene, a single-layer of carbon atoms arranged in a hexagonal lattice, with regards to its usage in future electronic devices; for instance in the context of quantum information processing. For a long time graphene was believed to be thermodynamically unstable. The discovery of this strictly two-dimensional material completed the family of carbon based structures, which had already been subject of intensive research with focus on zero-dimensional fullerenes and one-dimensional carbon nanotubes. Within only a few years of its discovery, the field of graphene related research has grown into one of today’s most diverse and prolific areas in condensed matter physics, highlighted by the award of the 2010 Nobel Prize in Physics to A.K. Geim and K. Noveselov for “their groundbreaking experiments regarding the two-dimensional material graphene”.
From the point of view of an experimental physicist interested in the electronic properties of a material system, the most intriguing characteristic of graphene is found in the Dirac-like nature of its charge carriers, a peculiar fact that distinguishes graphene from all other known standard semiconductors. The dynamics of charge carriers close to zero energy are described by a linear energy dispersion relation, as opposed to a parabolic one, which can be understood as a result of the underlying lattice symmetry causing them to behave like massless relativistic particles. This fundamentally different behavior can be expected to lead to the observation of completely new phenomena or the occurrence of deviations in well-known effects.
Following a brief introduction of the material system in chapter 2, we present our work studying the effect of induced superconductivity in mesoscopic graphene Josephson junctions by proximity to superconducting contacts in chapter 3. We explore the use of Nb as the superconducting material driven by the lack of high critical temperature and high critical magnetic field superconductor technology in graphene devices at that time. Characterization of sputter-deposited Nb films yield a critical transition temperature of \(T_{C}\sim 8{\rm \,mK}\). A prerequisite for successful device operation is a high interface quality between graphene and the superconductor. In this context we identify the use of an Ti as interfacial layer and incorporate its use by default in our lithography process. Overall we are able to increase the interface transparency to values as high as \(85\%\). With the prospect of interesting effects in the ballistic regime we try to enhance the electronic quality of our Josephson junction devices by substrate engineering, yet with limited success. We achieve moderate charge carrier mobilities of up to \(7000{\rm \,cm^2/Vs}\) on a graphene/Boron-nitride heterostructure (fabrication details are covered in chapter 5) putting the junction in the diffusive regime (\(L_{device}<L_{\rm{mfp}}\)). We speculate that either inhomogeneities in the graphene channel or lithography residues are responsible for this observation.
Furthermore we study the Josephson effect and Andreev reflection related physics in this device by low-temperature transport measurements. The junction carries a bipolar supercurrent which remains finite at the charge neutrality point. The genuine Josephson character is confirmed by the modulation of the supercurrent as a function of an out-of-plane magnetic field resembling that of a Fraunhofer-like pattern. This is further supported by the response of the junction to microwave radiation in the form of Shaprio steps. Surprisingly we find a strongly reduced superconducting energy gap of approximately \(\Delta = 400{\rm \,\mu eV}\) by quantitatively analyzing data of multiple Andreev reflections. We show this result to be consistent by careful analysis of the device parameters and comparison of these to a theoretical model. More experiments will be needed to determine the origin of this reduction and if the presence of the Ti interfacial layer plays an important role in that.
With regards to possible usability of superconducting contacts in more complex hybrid structures we can conclude that our work establishes the necessary preconditions while still leaving room for improvements; especially in terms of device quality.
In the second part of this work we are primarily interested in electrical transport properties of graphene nanodevices and their application in graphene-superconductor hybrid structures. The fact that graphene is mechanically stable down to a few tens of nanometers in width while exhibiting a finite conductance makes it an appealing choice as host for single-electron devices, also known as quantum dots. Our work on this topic is covered in chapter 4 where we first develop a high-resolution lithography process for the fabrication of single electron devices with critical feature sizes of roughly \(50{\rm \,nm}\). To this end we use a resist etch mask in combination with a reactive-ion etch process for device patterning. Carrier confinement in graphene is known to be hindered by the Klein tunneling phenomenon, a challenge that can be overcome by using all-graphene nano-constrictions to decouple the source and drain contacts from the central island.
The traditionally used constriction design is comprised of long and narrow connections. We argue that a design with very short and narrow constrictions could be beneficial for the quantum dot performance as the length merely affects the overall conductance and requires extended side-gates to control their transmission. We confirm the functionality of two different devices in low-temperature measurements, which differ in the size of their central island with \(d=250{\rm \,nm}\) for device no. 1 and \(d=400{\rm \,nm}\) for device no. 2. Coulomb blockade measurements conducted at \(20{\rm \,mK}\) on both devices reveal clear sequences of Coulomb peaks with amplitudes of up to \(0.8\rm{\,e}^2/\rm{h}\), a value significantly larger than what is commonly reported for similar devices. We interpret this as an indication of rather homogeneous constrictions, resulting from the modified design. Coulomb diamond measurements display the behavior expected for a lithographically designed single quantum dot revealing no features related to the presence of an additional dot. Using the stability diagram we determine the addition energies of the two dots and find them to be in good agreement with values reported in the literature for devices of similar size. Using the normalized Coulomb peak spacing as a figure of merit for the device quality we find that device no. 1 quantitatively compares well with a similar device fabricated on a superior hexagonal boron-nitride substrate. This result underlines the importance of non-substrate related extrinsic disorder sources and emphasizes the cleanliness of our lithography process.
Superconductor-graphene quantum dot hybrid structures employing Nb and Al electrodes were successfully fabricated from a lithography point of view, yet no evidence of any superconducting related effect was found in transport measurements. We assign the missing observation to interface issues that require careful analysis and likely a revision of the fabrication process.
A property equally important in graphene Josephson Junctions and quantum dots is the electronic quality of the device, as has been addressed in the previous paragraphs. It turns out that the \(\rm{SiO}_{2}\;\) substrate and lithography residues constitute the two major sources of disorder in graphene. In chapter 5 we present an approach based on the original work of Dean et al. who utilize hexagonal-Boron nitride as a replacement substrate for \(\rm{SiO}_{2}\). This idea was then extended by Wang et al. who also used this material as a shield to protect the graphene surface from contaminations during the lithography process. These structures are commonly referred to as van der Waals heterostructures and are assembled by stacking individual crystals on top of each other.
For this purpose we build a mechanical transfer system based on an optical microscope equipped with an additional micro-manipulator stage allowing precise alignment of two micrometer sized crystals with high precision. We demonstrate the functionality of this setup on the basis of successfully fabricated heterostructures. Furthermore a variation on the traditional method for single graphene/boron nitride structures is presented. Based on a reversed stacking order this method yields large areas of homogeneous graphene, however it comes with the drawback of limited yields. A common type of problem accompanying the fabrication of encapsulated graphene structures is the formation of contamination spots (also referred to as bubbles in the literature) at the interfaces between BN and graphene. We experience similar issues which we are unable to prevent and thus pose a limit to the maximum available device size. In the next step we develop a full lithography paradigm including high-resolution device patterning by electron beam lithography combined with reactive ion etching and two different ways to establish electrical contact to the encapsulated graphene flake. In this context we explore the use of three different types of etch masks and find a double layer of PMMA/HSQ best suited for our purposes. Our low power plasma etch process utilizes a combination of \(\rm{O}_{2}\;\) and \(\rm{CHF}_{3}\;\) and is optimized to show reproducible etch results.
A widely used method for electrical contacts relies on one-dimensional edge contacts whose functionality crucially depends on the use of Cr as the interface layer. For compatibility reasons with superconducting materials, e.g. Nb, we develop a self-aligned contact process that instead of only Cr is also compatible with Ti. We achieve this by modifying the plasma etch parameters such that the etch process exhibits extremely low graphene etch rates while keeping a high etch rate for h-BN. This allows clearing of a narrow stripe of graphene at the edge of the structure by using a thick PMMA layer as etch mask as replacement of the PMMA/HSQ combination. The purpose of this PMMA mask is two-fold since it also serves as lift-off mask during metalization.
The quality of the edge contacts fabricated with either method is excellent as determined from transport measurements at room and cryogenic temperatures. With typical contact resistances of a few hundred \({\rm \,}\Omega\mu{\rm m}\) and a record low of \(100{\rm \,}\Omega\mu{\rm m}\) the contacts can be considered to be state-of-the-art. The positive effect of encapsulation on the electronic quality is confirmed on a device exhibiting charge carrier mobilities exceeding \(10^5{\rm \,cm^2/Vs}\), one magnitude larger than what is commonly achieved on \(\rm{SiO}_{2}\).
The investigation of induced superconductivity in graphene Josephson Junctions, quantum dots, and high mobility heterostructures underlines the versatility of this material system, while covering only a tiny fraction of its prospects. Combination of the acquired knowledge regarding the physical effects and the developed lithography processes lay the foundation towards the fabrication and study of novel graphene hybrid devices.
Anxiety disorders (AD) are common, disabling mental disorders, which constitute the most prevalent mental health condition conveying a high individual and socioeconomic burden. Social anxiety disorder (SAD), i.e. fear in social situations particularly when subjectively scrutinized by others, is the second most common anxiety disorder with a life time prevalence of 10%. Panic disorder (PD) has a life time prevalence of 2-5% and is characterized by recurrent and abrupt surges of intense fear and anticipatory anxiety, i.e. panic attacks, occurring suddenly and unexpected without an apparent cue.
In recent years, psychiatric research increasingly focused on epigenetic mechanisms such as DNA methylation as a possible solution for the problem of the so-called “hidden heritability”, which conceptualizes the fact that the genetic risk variants identified so far only explain a small part of the estimated heritability of mental disorders.
In the first part of this thesis, oxytocin receptor (OXTR) gene methylation was investigated regarding its role in the pathogenesis of social anxiety disorder. In summary, OXTR methylation patterns were implicated in different phenotypes of social anxiety disorder on a categorical, neuropsychological, neuroendocrinological as well as on a neural network level. The results point towards a multilevel role of OXTR gene hypomethylation particularly at one CpG site (CpG3, Chr3: 8 809 437) within the protein coding region of the gene in SAD.
The second part of the thesis investigated monoamine oxidase A (MAOA) gene methylation regarding its role in the pathogenesis of panic disorder as well as – applying a psychotherapy-epigenetic approach – its dynamic regulation during the course of cognitive behavioural therapy (CBT) in PD patients. First, MAOA hypomethylation was shown to be associated with panic disorder as well as with panic disorder severity. Second, in patients responding to treatment MAOA hypomethylation was shown to be reversible up to the level of methylation in healthy controls after the course of CBT. This increase in MAOA methylation along with successful psychotherapeutic treatment was furthermore shown to be associated with symptom improvement regarding agoraphobic avoidance in an independent replication sample of non-medicated patients with PD.
Taken together, in the future the presently identified epigenetic patterns might contribute to establishing targeted preventive interventions and personalized treatment options for social anxiety disorder or panic disorder, respectively.
Functional and genetic dissection of mechanosensory organs of \(Drosophila\) \(melanogaster\)
(2016)
In Drosophila larvae and adults, chordotonal organs (chos) are highly versatile mechanosensors
that are essential for proprioception, touch sensation and hearing. Chos share molecular,
anatomical and functional properties with the inner ear hair cells of mammals. These multiple
similarities make chos powerful models for the molecular study of mechanosensation.
In the present study, I have developed a preparation to directly record from the sensory neurons
of larval chos (from the lateral chos or lch5) and managed to correlate defined mechanical inputs
with the corresponding electrical outputs. The findings of this setup are described in several case
studies.
(1) The basal functional lch5 parameters, including the time course of response during continuous
mechanical stimulation and the recovery time between successive bouts of stimulation, was
characterized.
(2) The calcium-independent receptor of α-latrotoxin (dCIRL/Latrophilin), an Adhesion class G
protein-coupled receptor (aGPCR), is identified as a modulator of the mechanical signals
perceived by lch5 neurons. The results indicate that dCIRL/Latrophilin is required for the
perception of external and internal mechanical stimuli and shapes the sensitivity of neuronal
mechanosensation.
(3) By combining this setup with optogenetics, I have confirmed that dCIRL modulates lch5
neuronal activity at the level of their receptor current (sensory encoding) rather than their ability
to generate action potentials.
(4) dCIRL´s structural properties (e.g. ectodomain length) are essential for the mechanosensitive
properties of chordotonal neurons.
(5) The versatility of chos also provides an opportunity to study multimodalities at multiple levels.
In this context, I performed an experiment to directly record neuronal activities at different
temperatures. The results show that both spontaneous and mechanically evoked activity increase
in proportion to temperature, suggesting that dCIRL is not required for thermosensation in chos.
These findings, from the development of an assay of sound/vibration sensation, to neuronal
signal processing, to molecular aspects of mechanosensory transduction, have provided the first
insights into the mechanosensitivity of dCIRL.
In addition to the functional screening of peripheral sensory neurons, another
electrophysiological approach was applied in the central nervous system: dCIRL may impact the
excitability of the motor neurons in the ventral nerve cord (VNC). In the second part of my work,
whole-cell patch clamp recordings of motor neuron somata demonstrated that action potential
firing in the dCirl\(^K\)\(^O\) did not differ from control samples, indicating comparable membrane
excitability.
Comparative transcriptomics and post-transcriptional regulation in \(Campylobacter\) \(jejuni\)
(2016)
The transcriptome is defined as the set of all RNA molecules transcribed in a cell. These include protein-coding messenger RNAs (mRNAs) as well as non-coding RNAs, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small non-coding RNAs (sRNAs). sRNAs are known to play an important role in regulating gene expression and virulence in pathogens. In this thesis, the transcriptome of the food-borne pathogen Campylobacter jejuni was characterized at single nucleotide resolution by use of next-generation sequencing approaches. The first genome of a C. jejuni strain was published in the year 2000. However, its transcriptome remained uncharacterized at large.
C. jejuni can survive in a variety of ecological niches and hosts. However, how strain-specific transcriptional changes contribute to such adaptation is not known. In this study, the global transcriptome maps of four closely related C. jejuni strains were defined using a differential RNA-seq (dRNA-seq) approach. This analysis also included a novel automated method to annotate the transcriptional start sites (TSS) at a genome-wide scale. Next, the transcriptomes of four strains were simultaneously mapped and compared by the use of a common coordinate system derived from whole-genome alignment, termed as SuperGenome. This approach helped to refine the promoter maps by comparison of TSS within strains. Most of the TSS were found to be conserved among all four strains, but some single-nucleotide-polymorphisms (SNPs) around promoter regions led to strain-specific transcriptional output. Most of these SNPs altered transcription only slightly, but some others led to a complete abrogation of transcription leading to differential molecular phenotypes. These in turn might help the strains to adapt to their specific host or microniche. The transcriptome also unveiled a plethora of sRNAs, some of which were conserved among the four strains while others were strain specific. Furthermore, a Cas9-dependent minimal type-II CRISPR-Cas system with only three Cas genes and multiple promoters to drive the transcription of the CRISPR locus was also characterized in C. jejuni using the dRNA-seq dataset.
Apart from sRNAs, the role of global RNA binding proteins (RBPs) is also unclear in C. jejuni. Aided by the global transcriptome data, the role of RBPs in post-transcriptional regulation of C. jejuni was studied at a global scale. Two of the most widely studied RNA binding proteins in bacteria are Hfq and CsrA. The RNA interactome of the translational regulator CsrA was defined using another global deep-sequencing technique that combines co-immunoprecipitation (coIP) with RNA sequencing (RIP-seq). Using this interactome dataset, the direct targets of this widespread global post-transcriptional regulator were defined, revealing a significant enrichment for mRNAs encoding genes involved in flagella biosynthesis. Unlike Gammaproteobacteria, where sRNAs such as CsrB/C, antagonize CsrA activity, no sRNAs were enriched in the CsrA-coIP in C. jejuni, indicating absence of any sRNA antagonists and novel modes of CsrA activity regulation. Instead, the CsrA regulatory pathway revealed flaA mRNA, encoding the major flagellin, as a dual-function mRNA. flaA mRNA was the main target of CsrA but it also served to antagonize CsrA activity along with the protein antagonist FliW previously identified in the Gram-positive bacterium Bacillus subtilis. Furthermore, this regulatory mRNA was also shown in this thesis to localize to the poles of elongating C. jejuni cells in a translation-dependent manner. It was also shown that this localization is dependent on the CsrA-FliW regulon, which controls the translation of flaA mRNA. The role and mechanism of flaA mRNA localization or mRNA localization in general is not yet clear in bacteria when compared to their eukaryotic counterparts.
Overall, this study provides first insights into riboregulation of the bacterial pathogen C. jejuni. The work presented in this thesis unveils several novel modes of riboregulation in C. jejuni, which could be applicable more generally. Moreover, this study also lays out several unsolved intriguing questions, which may pave the way for interesting studies to come.
microRNAs in chronic pain
(2016)
Chronic pain is a common problem in clinical practice, not well understood clinically, and frequently tough to satisfactorily diagnose. Because the pathophysiology is so complex, finding effective treatments for people with chronic pain has been overall less than successful and typically reduced to an unsatisfactory trial-and-error process, all of which translates into a significant burden to society. Knowledge of the mechanisms underlying the development of chronic pain, and moreover why some patients experience pain and others not, may aid in developing specific treatment regimens. Although nerve injuries are major contributors to pain chronification, they cannot explain the entire phenomenon. Considerable research has underscored the importance of the immune system for the development and maintenance of chronic pain, albeit the exact factors regulating inflammatory reactions remain unclear. Understanding the putative molecular and cellular regulator switches of inflammatory reactions will open novel opportunities for immune modulatory analgesics with putatively higher specificity and less adverse effects. It has become clear that small, non- coding RNA molecules known as microRNAs are in fact potent regulators of many thousands of genes and possibly cross-communicate between cellular pathways in multiple systems acting as so-called “master-switches”. Aberrant expression of miRNAs is now implicated in numerous disorders, including nerve injuries as well as in inflammatory processes. Moreover, compelling evidence supports the idea that miRNAs also regulate pain, and in analogy to the oncology field aid in the differential diagnosis of disease subtypes. In fact, first reports describing characteristic miRNA expression profiles in blood or cerebrospinal fluid of patients with distinct pain conditions are starting to emerge, however evidence linking specific miRNA expression profiles to specific pain disorders is still insufficient. The present thesis aimed at first, identifying specific miRNA signatures in two distinct chronic pain conditions, namely peripheral neuropathies of different etiologies and fibromyalgia syndrome. Second, it aimed at identifying miRNA profiles to better understand potential factors that differentiate painful from painless neuropathies and third, study the mechanistic role of miRNAs in the pathophysiology of pain, to pave the way for new druggable targets.
Three studies were conducted in order to identify miRNA expression signatures that are characteristic for the given chronic pain disorder. The first study measured expression of miR-21, miR-146a and miR-155 in white blood cells, skin and nerve biopsies of patients with peripheral neuropathies. It shows that peripheral neuropathies of different etiologies are associated with increased peripheral miR-21 and miR-146a, but decreased miR-155 expression. More importantly, it was shown that painful neuropathies have increased sural nerve miR-21 and miR-155 expression, but reduced miR-146a and miR-155 expression in distal skin of painful neuropathies. These results point towards the potential use of miRNAs profiles to stratify painful neuropathies. The seconds study extends these findings and first analyzed the role of miR-132-3p in patients and subsequently in an animal model of neuropathic pain. Interestingly, miR-132-3p was upregulated in white blood cells and sural nerve biopsies of patients with painful neuropathies and in animals after spared nerve injury. Pharmacologically modulating the expression of miR-132-3p dose-dependently reversed pain behavior and pain aversion, indicating the pro-nociceptive effect of miR-132-3p in chronic pain. This study thus demonstrates the potential analgesic impact by modulating miRNA expression. Fibromyalgia is associated with chronic widespread pain and, at least in a subgroup, impairment in small nerve fiber morphology and function. Interestingly, the disease probably comprises subgroups with different underlying pathomechanisms. In accordance with this notion, the third study shows that fibromyalgia is associated with both aberrant white blood cell and cutaneous miRNA expression. Being the first of its kind, this study identified miR-let-7d and its downstream target IGF-1R as potential culprit for impaired small nerve fiber homeostasis in a subset of patients with decreased intra-epidermal nerve fiber density. The work presented in this thesis is a substantial contribution towards the goal of better characterizing chronic pain based on miRNA expression signatures and thus pave the way for new druggable targets.